The long-term objective of the proposed research is to determine the cellular, subcellular and molecular actions of botulinum toxin. This objective is motivated by the need to understand the disease known as botulism and by the need to find ways to prevent and to treat botulism. Therefore, the focus of the work will be studies on cells of human origin, including cholinergic nerve cells (which are the target cells for toxin action in clinical poisoning), as well as fat cells and liver cells. Botulinum toxin acts on cholinergic nerve endings to block vesicle fusion and exocytosis. The toxin exerts this effect by proceeding through a sequence of events that involves binding to the plasma membrane, internalization, and eventual expression of an intracellular poisoning action. The final step is known to be enzymatic in nature; botulinum toxin is a zinc-dependent metalloendoprotease that cleaves peptide, that govern exocytosis. Recently, the applicant and his colleagues have found that botulinum toxin acts on other types of cells (e.g., fat, liver) to block other types of vesicle movement (e.g., vesicle shuttling, transcytosis). Therefore experiments will be done to determine whether a single sequence of events can account for toxin action on all vulnerable cells. Botulinum toxin is believed to act on nerve cells and other cells of laboratory animal origin by proteolytically cleaving four specific peptides (synaptobrevin, cellubrevin, SNAP-25 and syntaxin). However, it is not yet known whether these are the substrates that are the substrates that account for toxin action on human cells. Therefore, experiments will be done to identify and characterize human substrates for botulinum toxin. In keeping with the nature of clinical poisoning, initial emphasis will be on the study of human nerve cells. Subsequent experiments will be done on human fat cells and human liver cells. It is generally assumed that botulinum toxin action on vulnerable cells can be fully explained on the basis of cleavage of synaptobrevin, cellubrevin, SNAP-25 and syntaxin, but this has never been proved. Therefore, the applicant and his colleagues will use techniques that selectively remove each of these substances from target cells. The effects of poisoning with botulinum toxin will then be compared with the effects of selectively removing specific substrates. This will provide novel insights into toxin action, as well as expanding our knowledge of cell biology.